Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLbeta, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLbeta. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLbeta-dependent mechanism in non-proliferating cells and demonstrate that BER fidelity is lower in extracts from non-proliferating as compared with proliferating cells.